Induction Of Adhesion Molecule Expression Research Articles

Conditional over-expression of heme-oxygenase 1 in myelomonocytic cells reduces AngII-induced hypertension and vascular inflammation in mice

Abstract Background Systemic arterial Hypertension is one of the most important risk factor responsible for morbidity and mortality world-wide. Angiotensin II (Ang II), a potent vasoconstrictor and key player in the renin-angiotensin-aldosterone system (RAAS) is responsible for vascular dysfunction, inflammation, and tissue damage. Heme oxygenase-1 (HO-1) overexpression may confer antioxidant and anti-inflammatory properties in Ang II-induced hypertension through its action on heme catabolism, generating carbon monoxide (CO), ferritin and biliverdin/bilirubin by the action of biliverdin reductase A (BLVRA). Methods and results Male 9–12-weeks-old HO-1indxLySMCre/wt and LySMCre/wt mice were infused with AngII (1mgAngII/Day/Kg) for 7 days to induce hypertension and compared to sham treatment. Blood pressure monitoring by tail-cuff method, and endothelium-dependent vasodilator studies via organ chamber system using acetylcholine (ACh) in isolated aortic segments (~4 mm), revealed that overexpression of HO-1 in myeloid cells resulted in decreased systolic blood pressure and improved endothelial function in AngII-infused HO-1indxLySMCre/wt mice compared to controls. Concurrently, increased BLVRA expression in liver tissue and elevated plasma bilirubin levels were detected by transcriptome analysis and high-performance liquid chromatography, respectively. These results were supported by a reduction in the expression of inducible cytokines and cell adhesion molecules (CAMs) in the aorta, assessed using qPCR. Bone marrow transplant experiments demonstrated that bone marrow from LysMcre mice in HO-1indxLySMCre/wt mice abrogated the protective effect of HO-1, resulting in endothelial damage and increased blood pressure, potentially due to decreased liver BLVRA expression and the accumulation of bone marrow-derived cells in the vessel wall in response to AngII. Additionally, adeno-associated viral vector (AAV) transfection targeting specifically BLVRA activity in liver lead to an increase in blood pressure values and worse endothelium relaxation, in parallel with higher oxidative stress and the blood neutrophils concentration, as assessed in whole blood by using oxidative burst method and blood count system respectively. Conclusion The overexpression of HO-1 in myeloid cells confers anti-inflammatory protection in AngII-induced arterial hypertension in mice. Myeloid cells bone marrow-derived overexpressing HO-1 regulate the differentiation and infiltration of pro-inflammatory immune cells in the vasculature. BLVRA expression and bilirubin levels downstream of HO-1 play a critical role in protecting against vascular damage by reducing leukocyte infiltration.

Polystyrene microplastic particles induce endothelial activation.

Due to its increasing production, durability and multiple applications, plastic is a material we encounter every day. Small plastic particles from the μm to the mm range are classified as microplastics and produced for cosmetic and medical products, but are also a result of natural erosion and decomposition of macroplastics. Although being omnipresent in our environment and already detected in various organisms, less is known about the effects of microplastics on humans in general, or on vascular biology in particular. Here we investigated the effects of carboxylated polystyrene microplastic particles (PS, 1 μm) on murine endothelial and immune cells, which are both crucially involved in vascular inflammation, using in vitro and in vivo approaches. In vitro, PS induced adhesion molecule expression in endothelial cells with subsequent adhesion of leukocytes both under static and flow conditions. In monocytic cells, PS enhanced pro-inflammatory cytokine expression and release. Accordingly, administering mice with PS led to enhanced aortic expression of cytokines and adhesion molecules. Furthermore, we identified neutrophils as the PS-clearing blood leukocyte population. The findings from this study for the first time indicate polystyrene microplastic as a new environmental risk factor for endothelial inflammation.

Monocytes enhance the inflammatory response to TLR2 stimulation in aortic valve interstitial cells through paracrine up-regulation of TLR2 level.

Background and Objectives: Chronic valvular inflammation associated with monocyte infiltration promotes calcific aortic valve disease (CAVD) progression. Further, innate immunity in aortic valve interstitial cells (AVICs), mediated by Toll-like receptors (TLRs), up-regulates cellular inflammatory, fibrogenic and osteogenic activities. Currently, the pro-inflammatory communication between monocytes and AVICs and the underlying mechanism are unclear. We hypothesized that monocytes up-regulate AVIC inflammatory activity. This study sought to characterize the interaction between monocytes and AVICs and to elucidate the mechanism underlying cell-to-cell communication.Methods and Results: AVICs, monocytes and co-cultures were exposed to a low concentration of TLR2 activator Pam3CSK4 (0.03 µg/ml). The TLR2 activator at this dose induced a marked increase in AVIC production of ICAM-1 and VCAM-1 only when co-cultured with monocytes. Adding conditioned medium from Pam3CSK4-treated monocytes (Pam3 CM, containing 0.1 µg/ml of Pam3CSK4) to AVIC culture (30% vol/vol; diluting Pam3CSK4 to 0.03 µg/ml) greatly increased the expression of adhesion molecules while adding conditioned medium from untreated monocytes (control CM) had no effect. Inhibition or knockdown of TLR2 in AVICs markedly reduced ICAM-1 and VCAM-1 expression induced by Pam3 CM. Further, Pam3 CM increased TLR2 levels in AVICs. Multiplex-ELISA analysis of Pam3 CM identified greater levels of TNF-α. Neutralization of TNF-α abolished the effect of Pam3 CM on AVIC TLR2 levels, resulting in marked attenuation of its potency in the induction of adhesion molecule expression.Conclusions: This study demonstrates that activated monocytes use paracrine signaling to sensitize AVICs for inflammatory responses to a low level of TLR2 activator. The mechanism of sensitization involves up-regulation of AVIC TLR2 levels by TNF-α from monocytes. Infiltrated monocytes in aortic valve tissue may exacerbate valvular inflammation by rendering AVICs hypersensitive to TLR2 activators.

MON-214 1α,25-DIHYDROXYVITAMIN D3 TARGETING OF NF-ΚB SUPPRESSES TNF-Α INDUCED ADHESION MOLECULE EXPRESSION IN HUMAN ENDOTHELIAL CELLS

Vitamin D and its analogues have been documented to be associated with endothelial dysfunction in various diseases. However, the underlying mechanism remains unknown. Here, we conducted an in vitro study to evaluate the effect of 1α,25-dihydroxyvitamin D3, the active form of vitamin D3, on adhesion molecule expression in human endothelial cells. The possible mechanism involved in this process was also explored. Human umbilical vein cells (HUVECs) were cultured and treated according to the experiment requirement. Western Blot and RT-PCR were used to evaluate the expression of vascular cell adhesion molecular-1 (VCAM-1) and E-selectin. ChIP assay, immunofluorescence, Western Blot and co-immunoprecipitation were used to assess the effect of 1α,25-dihydroxyvitamin D3 on NF-κB signaling. 1α,25-dihydroxyvitamin D3 inhibited VCAM-1 and E-selectin mRNA and protein expression after TNF-α stimulation. ChIP assay showed that TNF-α increased the p65 binding to the promoter of VCAM-1 and E-selectin, which was suppressed by 1α,25-dihydroxyvitamin D3. 1α,25-dihydroxyvitamin D3 affected TNF-α induced IκBα phosphorylation and p65 NF-κB activation, leading to an inhibition of p65 nuclear translocation. These effects were reversed by a specific vitamin D receptor siRNA (VDR-siRNA). Co-immunoprecipitation revealed that 1α,25-dihydroxyvitamin D3 induced an increased binding of VDR to p65, which inhibited the ability of p65 binding to target gene promoters. 1,25-dihydroxyvitamin D3 suppresses TNF-α induced adhesion molecule expression in human endothelial cells by blocking the NF-κB pathway, and this was VDR dependent.

Administration of SGLT2 inhibitor empagliflozin against TNF‐α induced endothelial dysfunction in human venous and arterial endothelial cells.

IntroductionType 2 diabetes is commonly associated with vascular inflammation and endothelial dysfunction. Clinical trials have shown unexpected cardiovascular benefits of SGLT2 inhibitors (SGLT2i), a new class of glucose‐lowering agents, in diabetic individuals. We recently showed a direct cardiac class effect of SGLT2i; SGLT2i inhibit sodium hydrogen exchanger 1 (NHE‐1), reduce [Na+]c and induce vasodilation in the healthy heart. Here, we studied if the SGLT2i empagliflozin (EMPA) improves endothelial function during TNF‐α induced endothelial inflammation.MethodsHuman umbilical vein endothelial cells (HUVECs) and human coronary arterial endothelial cells (HCAECs) were pre‐incubated for 2h with either 1 μM, 3 μM EMPA or vehicle and subsequently exposed to 10 ng/mL TNF‐α in the presence of 1 μM (EMPA1), 3 μM EMPA (EMPA3) or vehicle. ROS levels (intracellular H2O2) were measured with a commercial kit and permeability was studied in a trans‐well assay using FITC‐albumin. VCAM‐1 and ICAM‐1 levels were determined using FACS analysis. Expression of (p)eNOS and caveolin1 was detected with western blot.ResultsEMPA treated HUVECs showed a tendency of reduced ROS after 48 h of TNF‐α stimulation (in nM; control 13.3±7.8, TNF 21.5±10.3 (p=0.067 vs. control) and TNF+EMPA1 16.5±6.2 (p=0.604 vs. control)). After 24 h, TNF‐α stimulation significantly increased permeability and EMPA administration did not restore this in HUVECs (albumin leakage in μg/mL: control 36.8±1.8, TNF 61.3±3.8, EMPA1 63.3±2.0). VCAM‐1 and ICAM‐1 levels were increased in both TNF and TNF+EMPA treated HUVECs after 4 h (TNF 6.4‐fold and 7.9‐fold, EMPA1 6.3‐fold and 7.6‐fold, EMPA3 5.7‐fold and 6.8‐fold respectively vs. control). Caveolin‐1 expression was 3.3‐fold increased after 6h of TNF‐α stimulation. Again, the presence of EMPA did not prevent this induction of adhesion molecule expression. Expression of peNOS/GAPDH and eNOS/GAPDH was downregulated after 24 h in both TNF (50% and 44% respectively) and EMPA1+TNF (60% and 49% respectively) conditions compared to control. Interestingly, HCAECs treated with TNF+EMPA showed recovered peNOS/GAPDH expression (TNF 69% and EMPA1 111% vs. control).ConclusionEMPA administration exhibited a trend of reduced ROS levels, but it did not affect permeability in HUVECs exposed to TNF‐α. (P)eNOS and caveolin expression remained unaffected in HUVECs, however (p)eNOS expression was recovered in HCAECs treated with EMPA. These findings may suggest endothelial cell type specific effects of EMPA during inflammation.Support or Funding InformationNone.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In vivo imaging of endothelial cell adhesion molecule expression after radiosurgery in an animal model of arteriovenous malformation.

Focussed radiosurgery may provide a means of inducing molecular changes on the luminal surface of diseased endothelium to allow targeted delivery of novel therapeutic compounds. We investigated the potential of ionizing radiation to induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (EC) in vitro and in vivo, to assess their suitability as vascular targets in irradiated arteriovenous malformations (AVMs). Cultured brain microvascular EC were irradiated by linear accelerator at single doses of 0, 5, 15 or 25 Gy and expression of ICAM-1 and VCAM-1 measured by qRT-PCR, Western, ELISA and immunocytochemistry. In vivo, near-infrared (NIR) fluorescence optical imaging using Xenolight 750-conjugated ICAM-1 or VCAM-1 antibodies examined luminal biodistribution over 84 days in a rat AVM model after Gamma Knife surgery at a single 15 Gy dose. ICAM-1 and VCAM-1 were minimally expressed on untreated EC in vitro. Doses of 15 and 25 Gy stimulated expression equally; 5 Gy was not different from the unirradiated. In vivo, normal vessels did not bind or retain the fluorescent probes, however binding was significant in AVM vessels. No additive increases in probe binding were found in response to radiosurgery at a dose of 15 Gy. In summary, radiation induces adhesion molecule expression in vitro but elevated baseline levels in AVM vessels precludes further induction in vivo. These molecules may be suitable targets in irradiated vessels without hemodynamic derangement, but not AVMs. These findings demonstrate the importance of using flow-modulated, pre-clinical animal models for validating candidate proteins for vascular targeting in irradiated AVMs.

Role of thrombin in the pathogenesis of central nervous system inflammatory diseases

Thrombin initiates proinflammatory signaling responses through activation of protease-activated receptors (PARs) in in vitro and in vivo systems. Proinflammatory signaling function of thrombin increases secretion of proinflammatory cytokines and chemokines, triggers vascular permeability, promotes leukocyte migration, and induces adhesion molecule expression. Thrombin as a potent signaling molecule is strongly implicated in a number of proinflammatory disorders including severe sepsis, cancer, cardiovascular disease, and of special interest in this review neurodegenerative disorders. This review summarizes the role of thrombin in the pathogenesis of central nervous system (CNS) inflammatory diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), promoting greater understanding and clinical management of these diseases. J. Cell. Physiol. 232: 482-485, 2017. © 2016 Wiley Periodicals, Inc.

Cryptotanshinone inhibits oxidized LDL-induced adhesion molecule expression via ROS dependent NF-κB pathways

ABSTRACTAdhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, play important roles in the initial stage of atherosclerosis. Cryptotanshinone (CPT), a natural compound isolated from Salvia miltiorrhiza Bunge, exhibits anti-atherosclerotic activity although the underlying mechanisms remain elusive. In this study, the protective effect of CPT against oxidized low-density lipoprotein (ox-LDL)-induced adhesion molecule expression was investigated in human umbilical vein endothelial cells. Ox-LDL significantly induced ICAM-1, VCAM-1, and E-selectin expression at the mRNA and protein levels but reduced eNOS phosphorylation and NO generation, which were reversed by CPT pretreatment. Sodium nitroprusside, a NO donor, N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, and BAY117082, a NF-κB inhibitor, inhibited ox-LDL-induced ICAM-1, VCAM-1, and E-selectin expression. Ox-LDL-induced ROS production was significantly inhibited by CPT and NAC. Furthermore, ox-LDL activated the NF-κB signaling pathway by inducing phosphorylation of IKKβ and IκBα, promoting the interaction of IKKβ and IκBα, and increasing p65 nuclear translocation, which were significantly inhibited by CPT. In addition, CPT, NAC, and BAY117082 inhibited ox-LDL-induced membrane expression of ICAM-1, VCAM-1, E-selectin, and endothelial–monocyte adhesion and restored eNOS phosphorylation and NO generation. Results suggested that CPT inhibited ox-LDL-induced adhesion molecule expression by decreasing ROS and inhibiting the NF-κB pathways, which provides new insight into the anti-atherosclerotic mechanism of CPT.

Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation.

Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary human macro- and microvascular ECs treatment with monoclonal pan- and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation.

Abstract 16665: Caveolin-1 and α7nachr Interaction is Essential in Preventing Nicotine Induced Vascular Inflammatory Response in Mice

Nicotine is an independent risk factor of and associated with the development of atherosclerosis. Dysfunction of endothelial Cavolin-1 (Cav-1) can accelerate atherosclerosis progression via boosting inflammation response. However, the causative link between nicotine and atherosclerosis remains unknown. Here, we utilized molecular and cellular experiments revealed that in human endothelial cells (HUVECs), 10-6 M nicotine increased intracellular oxygen free radical generation, inhibited eNOS activity, augmented pathological NO production from iNOS, promoted nitrative stress (Nitrotyrosine), and increased adhesion molecule expression when compared with vehicle. In vivo administration of nicotine upregulated plasma adhesion molecule levels and significantly increased nitrotyrosine levels in vascular tissue (2±0.4 fold compared with untreated group, p<0.05). Bioinformatics analysis predicted that α7nAChR motif (151YIDVRWFPF159 and 168FGSWSYGGW176) may interact with Cav-1 via Cav-1 scaffolding domain (82DGIWKASFTTFTVTKYWFYR101). The bioinformatic analysis was confirmed by immunocytochemistry and immunoprecipitation showing that α7nAChR co-localizes with caveolin-1 (Cav-1) and these two molecules interact reciprocally. Although 10-6M nicotine did not alter expression levels of Cav-1, it significantly decreased the interaction between Cav-1 and nicotine receptor (α7nAchR). After siRNA Cav-1 gene silence, nicotine treatment further significantly increased ROS generation (1.37 + 0.33 times vs. scrambled siRNA, p<0.05), enhanced pathological NO production (1.35 ± 0.4 fold, p<0.05), and increased adhesion molecule expression (1.4 ±0.4 fold, p<0.05). AMPKα1, a sensor of oxidative stress, was phosphorylated after nicotine treatment. However, siRNA AMPK gene silence failed to block nicotine induced adhesion molecule expression. Taken together, these results demonstrated that reduced α7nAchR/Cav-1interaction plays an important role in nicotine induced endothelial inflammatory response; this inflammation signal pathway is AMPK independent. Preserving and strengthening stability of Cav- 1/α7nAchR interaction is a potential target in treatment of vascular dysfunction caused by smoking.

Palmitate-stimulated monocytes induce adhesion molecule expression in endothelial cells via IL-1 signaling pathway.

Increased intake of saturated fatty acids (SFAs), such as palmitate (Pal), is linked to a higher risk of type 2 diabetes and cardiovascular disease. Although recent studies have investigated the direct effects of SFAs on inflammatory responses in vascular endothelial cells, it remains unknown whether SFAs also induce these responses mediated by circulating cells. In this study, especially focused on adhesion molecules and monocytes, we investigated the indirect effects of Pal on expression and release of ICAM-1 and E-selectin in vascular endothelial cells. Phorbol 12-myristate 13-acetate (PMA)-treated THP-1 (pTHP-1) cells and human monocytes were stimulated with various free fatty acids (FFAs). SFAs, but not unsaturated fatty acids (UFAs), increased interleukin (IL)-1β secretion and decreased IL-1 receptor antagonist (IL-1Ra) secretion, resulting in an increase in the IL-1β/IL-1Ra secretion ratio. UFAs dose-dependently inhibited the increase in IL-1β secretion and decrease in IL-1Ra secretion induced by Pal. Moreover, in human aortic and vein endothelial cells, expression and release of ICAM-1 and E-selectin were induced by treatment with conditioned medium collected from Pal-stimulated pTHP-1 cells and human monocytes, but not by Pal itself. The up-regulated expression and release of adhesion molecules by the conditioned medium were mostly abolished by recombinant human IL-1Ra supplementation. These results suggest that the Pal-induced increase in the ratio of IL-1β/IL-1Ra secretion in monocytes up-regulates endothelial adhesion molecules, which could enhance leukocyte adhesion to endothelium. This study provides further evidence that IL-1β neutralization through receptor antagonism may be useful for preventing the onset and development of cardiovascular disease.

Abstract 241: Anti-Inflammatory Properties of Cynomolgus Monkey and Rabbit High-Density Lipoprotein Following Treatment With Recombinant Lecithin Cholesterol Acyltransferase

Introduction: Lecithin cholesterol acyltransferase (LCAT) modulates HDL formation and increases HDL-C. Anti-inflammatory properties of HDL may be atheroprotective. We evaluated HDL from rabbits and cynomolgus monkeys treated with recombinant LCAT (rLCAT) to determine its anti-inflammatory properties. Methods: HDL from serum was isolated by ultracentrifugation. HUVECs were treated with HDL, stimulated with TNFα, and mRNA expression of VCAM-1, E-Selectin, and ICAM-1 was measured. Results: TNFα induced adhesion molecule expression in HUVECs treated with HDL (0.3 mg/mL) from rLCAT treated rabbits was reduced (E-Selectin) or similar (VCAM-1, ICAM-1) compared to pre-dose HDL. Using HDL (0.3 mg/mL) from rLCAT treated cynomolgus monkeys reduced expression of E-Selectin, VCAM-1, and ICAM-1 compared to control (pre-dose or vehicle) HDL in a rLCAT dose-dependent manner. In HUVECs treated with cynomolgus monkey HDL at concentrations normalized to serum HDL-C, adhesion molecule expression was reduced but showed a different profile. HDL after treatment with 1 or 5 mpk rLCAT reduced expression more than HDL from controls, while HDL after treatment with 20 or 150 mpk rLCAT blocked expression less than controls. Conclusions: HDL from rabbits and cynomolgus monkeys after rLCAT treatment was equally or more anti-inflammatory compared to control HDL when tested at a fixed concentration, a measure of HDL quality. To account for treatment changes to both HDL quality and quantity, HDL was tested relative to serum HDL-C. In this case, HDL from the 1 mpk group proved most efficacious in reducing TNFα stimulated adhesion molecule expression compared to vehicle HDL or HDL from higher doses.

Increased Platelet-Derived CD40L May Modulate Endothelial Cell ICAM-1 Expression and Interact With Leukocytes In Sickle Cell Anemia

BackgroundVaso-occlusive events are a major cause of morbidity in sickle cell anemia (SCA) and attributable to the abnormal adhesion of red cells and leukocytes to the endothelium. Platelets may contribute to the chronic inflammation and endothelial activation that initiates the vaso-occlusive process. We hypothesized that platelet-associated CD40 ligand (CD40L) may contribute to platelet-mediated inflammatory responses in SCA. AimsThis study evaluated the platelet (PLT) release of CD40L, the expression of its receptor (CD40) on platelets, neutrophils, lymphocytes and monocytes of control individuals (CON) and SCA patients, and also the ability of platelet-derived CD40L to activate endothelial cells. MethodsIL-8, soluble ICAM-1, VCAM-1 and CD40L were determined in PLT-free plasma or the supernatant of stimulated (ADP or Collagen) and unstimulated PLTs (2•10⁸/mL in Kreb's buffer), from CON individuals and steady-state SCA patients, by ELISA. Flow cytometry was used to analyze CD40 expression on platelets, neutrophils, lymphocytes and monocytes from the peripheral blood of the study's subjects. Human umbilical vein endothelial cells (HUVECs) were cultured (1x106cells/well; 37°C, 5% CO2) together with PLTs (3x108PLTs/well) from CON individuals or steady-state SCA patients for 24h, 37°C, 5%CO2, in the presence, or not, of blocking antibodies against CD40L. After incubation, PLTs were removed and HUVECs analyzed by flow cytometry for CD54 (ICAM-1) surface expression. ResultsSCA individuals presented elevated levels of plasma CD40L (724.4± 55.7 pg/ml; n=90) compared to CON (241.5±34.6 pg/ml; n=41; P<0.0001) and these levels correlated with PLT counts (rs=0.255; P=0.015). No correlation was found between plasma CD40L and plasma IL-8, ICAM-1 or VCAM-1. PLT release of CD40L (90 min, 37°C, 5%CO2) was evaluated; PLTs of SCA patients released higher quantities of CD40L (8347±1464 pg/108 PLTs; n=10) than PLTs of CON individuals (3652±568 pg/108 PLTs; n=5; P=0.019). CD40L release from SCA PLTs was augmented by incubation with collagen (P<0.001), but not ADP. Expression of the CD40 receptor on the platelet surface was elevated in the SCA group (52.4±2.7% positive cells; n=23), compared to the CON group (36.8±3.7% positive cells; n=9; P=0.005). The surface expression of CD40 was also elevated on neutrophils (SCA, 10.4±1.5% positive cells, n=14; CON, 5.5±1.1% positive cells, n=13; P=0.03), lymphocytes (SCA, 8.3±0.8% positive cells, n=16; CON, 3.6±0.4% positive cells, n=14; P<0.001) and monocytes (SCA 69.6±5.9% positive cells, n=16; CON, 49.9±5.8% positive cells, n=14; P=0.03) of SCA patients, compared to controls. ICAM-1 expression on the surface of HUVECs (Basal expression 32.8±1.8%, n=11) was significantly increased following incubation with SCA PLTs (54.0±4.8%, n=11, p<0.0001) and slightly augmented after incubation with CON PLTs (40.8±3.1%, n=11, p<0.05; Repeated measures ANOVA). Interestingly, when HUVECs and SCA PLTs were incubated with a blocking antibody against CD40L, the increase in ICAM-1 expression was significantly reversed on HUVECs (HUVECs, 28.1±0.2%, n=6; HUVECs+SCA PLTs, 42.0±3.3%, n=6; HUVECs+SCA PLTs+anti-CD40L 28.9±1.5%, n=6; P<0.01). ConclusionsPlasma levels and platelet release of CD40L were found to be significantly elevated in SCA, in association with increased expressions of the CD40 receptor on SCA PLTs, neutrophils, lymphocytes and monocytes, possibly indicating a CD40L-mediated crosstalk between platelets and leukocytes in SCA. Platelets from SCA patients can induce adhesion molecule expression on the surface of endothelial cells in vitro, and this up-regulation may be modulated by platelet-derived CD40L. Results suggest that the CD40/CD40L pathway may be altered in SCA and that platelets may participate in this up-regulation. Given the potent inflammatory effect of this cytokine, a role for platelets and this cytokine in endothelial activation, inflammation and consequent vaso-occlusion, is likely. Disclosures:No relevant conflicts of interest to declare.

Induction of adhesion molecule expression in co-culture of human bronchial epithelial cells and neutrophils suppressed by puerarin via down-regulating p38 mitogen-activated protein kinase and nuclear factor κB pathways

In this study, we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system, assess the effects of puerarin on suppressing these adhesion molecules expressions, and explore the roles of two crucial signal-transduction elements p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappa B (NF-κB) in modulating adhesion molecules expressions. Neutrophils and BEAS-2B cells (one human bronchial epithelial cell line) were co-cultured, and adhesion molecules expressions on cell surface were detected using flow cytometry. The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction (real-time qPCR). Phosphorylated p38 MAPK and inhibitor κB were analyzed by Western blot. In co-culture system, adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly (P<0.05). Correspondingly, the mRNA levels of adhesion molecules were also increased greatly. Moreover, the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface. Furthermore, phosphorylated p38 MAPK and inhibitor κB in BEAS-2B cells and neutrophils were elevated in co-culture system, but decreased significantly after upon the treatment of peurarin (P<0.05). Coculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflflammation, whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of p38 MAPK and NF-κB pathways, and exhibiting its anti-inflflammation activity.

Directing TRAF-ic

The recognition that atherosclerosis is a chronic inflammatory disease has emphasized the fundamental link between the innate immune system and disease pathogenesis. Atherosclerotic lesions develop at vulnerable locations throughout the vasculature as a result of detection of endogenous or microbial ligands by germ-line encoded pattern recognition receptors, including the innate immune Toll-like receptors (TLRs).1,2 Certain dietary lipids that contribute to atherosclerotic disease resemble on a molecular level lipids expressed in the microbial cell wall and are recognized by TLRs. Engagement via TLRs of dietary lipid molecules on both immune and nonimmune cells initiates inflammatory responses that persist chronically and culminate in the deposition of fatty streaks, which progress to occlusive atherosclerotic plaques.3 Article see p 1739 The vascular endothelium is central in the initial cellular and molecular responses to not only infection and tissue injury, but also to overly abundant dietary lipids. Vascular endothelial cells express TLRs, and a fundamental event in the initiation of atherosclerosis is the upregulation of adhesion molecules on endothelial cells that promote localized and restricted recruitment of circulating leukocytes and platelets that support leukocyte tethering and rolling, firm adhesion, and transmigration into underlying tissues. This amassing of cells contributes to the composition of the atherosclerotic lesion. In addition to the induction of adhesion molecule expression, dietary lipids can also incite the production of inflammatory cytokines by endothelial cells, which act in an autocrine and paracrine manner to feed the inflammatory response. Innate immune activation of hematopoietic cells, most notably monocytes and macrophages, is another central response that leads to the development of chronic atherosclerotic disease. Monocytes and macrophages express TLRs that respond to microbes and dietary lipids in much the same manner as endothelial cells, although the outcome of TLR signaling differs as a result of cell-specific functions. Activated macrophages secrete …

THE PROTECTIVE EFFECTS OF X-BOX BINDING PROTEIN 1 ON TUMOUR NECROSIS FACTOR-ALPHA INDUCED PRO-INFLAMMATORY RESPONSE

ObjectivesTumour necrosis factor alpha (TNF-α) is a potent pro-inflammatory factor playing a critical role in the initiation and progression of atherosclerosis. Exposure of vascular endothelial cells to TNF-α is known...

Abstract 222: D-Series Resolvins Modulate Postinjury Signaling Pathways in Vascular Smooth Muscle Cells

Introduction: Recent work suggests that the resolution of inflammation is an active process involving specialized molecules that are locally generated. D-series resolvins (RvD) are endogenous pro-resolving lipid mediators derived from the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA). VSMC activation following vascular injury leads to neointimal hyperplasia which may reflect a relative “resolution deficit” in the vessel wall. Objectives: We sought to determine the effects of RvD on injury-related inflammatory and growth factor signaling pathways in VSMC, using in vitro and in vivo models. Hypothesis: We hypothesize that RvD will attenuate VSMC activation responses and modulate vascular injury. Methods: Human VSMC were treated with cytokines (TNF-α, IL-1β), growth factors (PDGF, AngII) or oxidant stress (H 2 O 2 ) in the presence of RvD1 or RvD2 (.01-100 nM), and phenotype was assessed by cytotoxicity, proliferation, migration (transwell), monocyte adhesion (U937) and adhesion molecule expression (qPCR and flow cytometry) assays. NZW rabbits (N=4) underwent bilateral femoral balloon injury with intraluminal delivery of RvD2 (10 nM vs vehicle control) to examine acute effects (3 days) on the injury response. Results: RvD treatment of VSMC produced dose-dependent inhibition of VSMC proliferation (max %inhibition: RvD1 58.8%, p&lt;0.01, RvD2 63.1%, p&lt;0.01), migration (RvD1 66.1%, p&lt;0.01%, RvD2 68.8%, p&lt;0.01), monocyte adhesion (RvD1 36.0%, p&lt;0.01, RvD2 41.0%, p&lt;0.01), and inducible adhesion molecule expression (VCAM-1: RvD1 70.5%, p&lt;0.01, RvD2 77.2%, p&lt;0.01; ICAM-1: RvD1 65.4%, p&lt;0.01, RvD2 72.1%, p&lt;0.01) without measurable cytotoxicity. In the rabbit model of balloon arterial injury, proliferation was significantly reduced at 3 days in RvD2 treated vessels (%inhibition 57%, p&lt;0.01). Conclusion: RvD broadly inhibit VSMC activation responses and may modulate the vascular injury response in-vivo .

γ-Oryzanol Reduces Adhesion Molecule Expression in Vascular Endothelial Cells via Suppression of Nuclear Factor-κB Activation

γ-Oryzanol (γ-ORZ) is a mixture of phytosteryl ferulates purified from rice bran oil. In this study, we examined whether γ-ORZ represents a suppressive effect on the lipopolysaccharide (LPS)-induced adhesion molecule expression on vascular endothelium. Treatment with LPS elevated the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin in bovine aortic endothelial cells (BAECs). Pretreatment with γ-ORZ dose-dependently decreased the LPS-mediated expression of these genes. Western blotting also revealed that pretreatment with γ-ORZ dose-dependently inhibited LPS-induced VCAM-1 expression in human umbilical vein endothelial cells. Consistently, pretreatment with γ-ORZ dose-dependently reduced LPS-induced U937 monocyte adhesion to BAECs. In immunofluorescence, LPS caused nuclear factor-κB (NF-κB) nuclear translocation in 40% of BAECs, which indicates NF-κB activation. Pretreatment with γ-ORZ, as well as its components (cycloartenyl ferulate, ferulic acid, or cycloartenol), dose-dependently inhibited LPS-mediated NF-κB activation. Collectively, our results suggested that γ-ORZ reduced LPS-mediated adhesion molecule expression through NF-κB inhibition in vascular endothelium.

P1-12-08: Induction of Adhesion Molecules on Target Cells Maximizes Antibody-Dependent Cell-Mediated Cytotoxicity of Anti-Her2 Therapeutic Antibody with High Fucose Glycan.

Abstract Background: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one pivotal effector function of certain therapeutic antibodies. ADCC is the lysis of antibody-coated target cells by effector cells expressing Fc receptors (FcR) that recognize the fragment crystallizable (Fc) region of the bound antibody. Higher fucosylated terminal glycan on Fc results in significantly lower ADCC activity because core fucose sterically hinders the binding of Fc region to FcR on effector cells. The role of adhesion molecules in ADCC is unclear but it has been reported that adhesion molecules help better orientation of effector cells (natural killer cells) toward adhesion molecule-coated beads. We hypothesize that a preferable association of effector cells and target cells mediated by adhesion molecules may compensate ADCC loss due to the steric hindrance of high core fucose on Fc. Recombinant humanized monoclonal anti-Her2 antibodies (anti-Her2 rhuMAb) at various fucosylation levels have been used in this study to evaluate the role of adhesion molecules in ADCC of antibodies with high fucose glycan. Materials and Methods: Natural killer (NK) cell line transfected with FcR were used as effector cells. Her2-expressing breast cancer cell lines BT-474 and SK-BR3 were used as target cells. Adhesion molecules were induced in target cells by incubation with 50ng/ml of recombinant human tumor necrosis factor (TNFa) for 1–2 days. Anti-Her2 rhuMAbs with different Fc fucosylation (G0-F from &amp;lt;1% to &amp;gt;2%) were added in the co-culture of effector cells and target cells. ADCC activities were quantitated by target cells lysis assessment using Europium-TDA release assay. Results: Untreated BT-474 or SK-BR3 target cells had very little adhesion molecule expression. A high fucosylated anti-Her2 rhuMAb induced 52% of target cell lysis, which represented two-thirds of cell lysis by a low fucosylated Ab. Increment of E:T ratio could not maximize ADCC activities. With TNFa treatment, target cell lysis was increased from 52% to 100% by high fucose rhuMAb. This enhancement of ADCC activities is probably due to the expression of adhesion molecules such as ICAM-1 and VCAM-1, which was shown to be up-regulated by 100-fold on target cells in the presence of TNFa. Our data suggested that ADCC activities in Her2-expressing breast cancer cell lines were HER2−specific, anti-HER2 rhuMAb-dependent and adhesion molecules were indispensible for the enhanced ADCC activities. Discussion: rhuMAbs produced in Chinese hamster ovary (CHO) cell lines may contain various Fc fucosylation, hence various ADCC activities. Although up-regulation of adhesion molecules alone cannot lyse target cells directly, our data suggested maximized ADCC activities by rhuMAbs with high Fc fucosylation level could be achieved by induction of adhesion molecule expression on target cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-12-08.

DEHP and its active metabolites: leaching from different tubing types, impact on proinflammatory cytokines and adhesion molecule expression. Is there a subsumable context?

Di(2-ethylhexyl)phthalate (DEHP) is suspected to be toxic for several reasons. During contact with a lipophilic medium, DEHP leaks from polyvinylchloride (PVC), but its influence on inflammatory reactions remains unknown. We examined specific DEHP leaching out of different tubing types, the possibly modulated liberation of proinflammatory cytokines and the induction of adhesion molecule expression in primary endothelial cells. Blood samples were circulated in traditional PVC, nodioctyl phthalate (DOP) PVC and heparin-coated PVC tubing within a Chandler loop model. The blood was tested for the concentration of DEHP and its active metabolites as well as the liberation of the proinflammatory cytokines TNFα and IL1ß. Furthermore, we exposed human endothelial cells to circulated blood and analysed them for the expression of the adhesion molecules ICAM-1, VCAM-1 and E-selectin. In contrast to the other tubing, PVC tubing showed significantly elevated DEHP levels, but no alteration was observed concerning a potential up-regulation of the cytokines or activation of the endothelial adhesion molecule receptors. Our data conclude that there is no correlation between DEHP leaching and the inflammatory response after ECC support, but this study showed that even DEHP-free material is leaching DEHP and its toxic metabolites.